Muscle hypertrophy involves the activation and incorporation of satellite cell nuclei into existing muscle fibres, to keep the myonuclear domain relatively constant (1). However, it is not known whether accretion of myonuclei precedes or follows the increase in fibre cross-sectional area and whether this time-course is affected by age. The left plantaris muscle of 5- and 25-month-old male Wistar rats was overloaded by denervation of its synergists for 1, 2 or 4 weeks (n=3-5) with the contra-lateral plantaris muscle serving as control. All surgeries were carried out under isoflurane anaesthesia and aseptic conditions. During the terminal experiment the animals were killed by an intraperitoneal injection of an overdose of pentobarbital sodium. Myonuclei were counted in haematoxylin-stained cross-sections. Data were analysed with ANOVA. Myonuclear domain of type I was smaller than that of type II fibres (p = 0.001). Moreover, the myonuclear domain of type II fibres was larger in the glyocolytic than in the oxidative regions of the muscle (p = 0.007). This indicates that the number of myonuclei per muscle fibre is not only determined by size, but also by fibre type and location of the fibre in the muscle. The proportion of central nuclei was approximately ten times higher in old than in young adult muscles irrespective of overload (p < 0.001). While muscle hypertrophy became significant two weeks after overload (33% in young adult; p = 0.008), the increase in myonuclear number was significant only at four weeks of overload (p < 0.0001). The time course and magnitude of hypertrophy was similar in young adult and old rats. In conclusion, our data indicate that acquisition of new myonuclei is not necessary for the initial hypertrophy. In addition, despite indications in the literature that muscle regeneration is impaired in 25-month-old rats our study shows that the ability to develop hypertrophy was not attenuated or delayed at this age.